LPAAT-B inhibitors and uses thereof

ABSTRACT

The invention relates to triazines and the use thereof to inhibit lysophosphatidic acid acyltransferase β (LPAAT-β) activity. The invention further relates to methods of treating cancer using said triazines. The invention also relates to methods for screening for LPAAT-β activity.

CROSS-REFERENCE INFORMATION

[0001] This application claims priority under 35 U.S.C. 119(e) to U.S.Provisional Application Serial No. 60/244,195, filed Oct. 31, 2000, thedisclosure of which are incorporated by reference herein in itsentirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention is in the field of organic and medicinal chemistry.In particular, the invention relates to triazines and the use thereof toinhibit lysophosphatidic acid acyltransferase β (LPAAT-β) activity. Theinvention further relates to methods of treating cancer using saidtriazines. The invention also relates to methods for screening forLPAAT-β activity.

[0004] 2. Related Art

[0005] LPAAT catalyzes the acylation of lysophosphatidic acid (LPA) tophosphatidic acid. LPA is the simplest glycerophospholipid, consistingof a glycerol molecule, a phosphate group, and a fatty acyl chain. LPAATadds a second fatty acyl chain to LPA, producing phosphatidic acid (PA).PA is the precursor molecule for certain phosphoglycerides, such asphosphatidylinositol, and diacylglycerols, which are necessary for theproduction of other phosphoglycerides, such as phosphatidylcholine, andfor triacylglycerols, which are essential biological fuel molecules.

[0006] In addition to being a crucial precursor molecule in biosyntheticreactions, LPA has recently been added to the list of intercellularlipid messenger molecules. LPA interacts with G protein-coupledreceptors, coupling to various independent effector pathways includinginhibition of adenylate cyclase, stimulation of phospholipase C,activation of MAP kinases, and activation of the small GTP-bindingproteins Ras and Rho. Moolenaar, J. Biol. Chem 28:1294 (1995). Thephysiological effects of LPA have not been fully characterized as yet.However, one of the physiological effects that is known is that LPApromotes the growth and invasion of tumor cells. It has been shown thatthe addition of LPA to ovarian or breast cancer cell lines induces cellproliferation, increases intracellular calcium levels, and activates MAPkinase. Xu et al., Biochem. J. 309:933 (1995). In addition, LPA has beenshown to induce MM1 tumor cells to invade cultured mesothelial cellmonolayers. Imamura et al. Biochem. Biophys. Res. Comm. 193:497 (1993).

[0007] Like LPA, PA is also a messenger molecule. PA is a key messengerin a common signaling pathway activated by proinflammatory mediatorssuch as interleukin-1β, tumor necrosis factor α, platelet activatingfactor, and lipid A. Bursten et al., Am. J. Physiol. 262:C328 (1992);Bursten et al., J. Biol. Chem. 255:20732 (1991); Kester J. Cell Physiol.156:317 (1993). PA has been implicated in mitogenesis of several celllines [English, Cell Signal 8: 341 (1996)]. PA level has been found tobe increased in either ras or fps transformed cell lines compared to theparental Rat2 fibroblast cell line [Martin et al., Oncogene 14: 1571(1997)]. Activation of Raf-1, an essential component of the MAPKsignaling cascade, by extracellular signals is initiated by associationwith intracellular membranes. Recruitment of Raf-1 to membranes has beenreported to be mediated by direct association with phosphatidic acid[Rizzo et al., J Biol Chem 275:23911-8 (2000)]. Thus, LPAAT, as anenzyme that regulate PA content in cells, may play a role in cancer, andmay also mediate inflammatory responses to various proinflammatoryagents.

SUMMARY OF THE INVENTION

[0008] The preferred embodiments of the present invention relate to acompound of the Formula:

[0009] wherein,

[0010] R¹ is halo, hydroxy, alkylmercapto, mercapto, alkoxy, aryloxy orsubstituted amino;

[0011] R², R³, R⁴ and R⁵, each of which may be same or different, arehydrogen, alkyl, substituted alkyl, alkenyl, alkynyl, aryl orsubstituted aryl; or

[0012] R² and R³ or R⁴ and R⁵, together with the nitrogen to which theyare attached, form a piperidine, piperazine, or a morpholine ring; or

[0013] pharmaceutically acceptable salts thereof.

[0014] The preferred embodiments of the present invention further relateto a method for inhibiting LPAAT-β (lysophosphatidic acidacyltransferase β) comprising contacting LPAAT-β with an effectiveamount of a compound of the Formula:

[0015] wherein,

[0016] R¹ is halo, hydroxy, alkylmercapto, mercapto, alkoxy, aryloxy orsubstituted amino;

[0017] R², R³, R⁴ and R⁵, each of which may be same or different, arehydrogen, alkyl, substituted alkyl, alkenyl, alkynyl, aryl orsubstituted aryl; or

[0018] R² and R³ or R⁴ and R⁵, together with the nitrogen to which theyare attached, form a piperidine, piperazine, or a morpholine ring; or

[0019] pharmaceutically acceptable salts thereof;

[0020] thereby inhibiting LPAAT-β.

[0021] The preferred embodiments of the present invention further relateto a method of inhibiting cell proliferation comprising contacting acell with an effective amount of a compound of the Formula:

[0022] wherein,

[0023] R¹ is halo, hydroxy, alkylmercapto, mercapto, alkoxy, arylox orsubstituted amino;

[0024] R², R³, R⁴ and R⁵, each of which may be same or different, arehydrogen, alkyl, substituted alkyl, alkenyl, alkynyl, aryl orsubstituted aryl; or

[0025] R² and R³ or R⁴ and R⁵, together with the nitrogen to which theyare attached, form a piperidine, piperazine, or a morpholine ring; or

[0026] pharmaceutically acceptable salts thereof;

[0027] thereby inhibiting the proliferation of the cell.

[0028] The preferred embodiments of the present invention further relateto a method for treating cancer, comprising administering to an animalin need thereof, an effective amount of a compound of the Formula:

[0029] wherein,

[0030] R¹ is halo, hydroxy, alkylmercapto, mercapto, alkoxy, aryloxy orsubstituted amino;

[0031] R², R³, R⁴ and R⁵, each of which may be same or different, arehydrogen, alkyl, substituted alkyl, alkenyl, alkynyl, aryl orsubstituted aryl; or

[0032] R² and R³ or R⁴ and R⁵, together with the nitrogen to which theyare attached, form a piperidine, piperazine, or a morpholine ring; or

[0033] pharmaceutically acceptable salts thereof;

[0034] wherein the cancer is treated.

[0035] The preferred embodiments of the present invention further relateto a method for screening a patient for LPAAT-β activity, said methodcomprising detecting the presence or absence of an increased amount ofLPAAT-β RNA, DNA or protein relative to a predetermined control, wherebythe presence of said increased amount is indicative of cancersusceptibility in said patient.

[0036] The preferred embodiments of the present invention further relateto a method of inhibiting cell proliferation comprising the inhibitionof LPAAT-β.

[0037] The preferred embodiments of the present invention further relateto a vaccine preparation capable of inducing an anti-tumor immuneresponse comprising a pharmaceutically acceptable carrier and ananti-tumor immune response-inducing effective amount of LPAAT-β protein.

[0038] The preferred embodiments of the present invention further relateto a method for screening a patient for LPAAT-β activity, said methodcomprising detecting the presence or absence of an increased amount of aphospholipid of defined acyl-chain composition relative to apredetermined control, whereby the presence of said increased amount isindicative of cancer susceptibility in said patient.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039]FIG. 1 shows breast intraductal adenocarcinoma samples.

[0040]FIG. 2 shows intraductal adenocarcinoma samples.

[0041]FIG. 3 shows three examples of ovarian cancer samples.

[0042]FIG. 4A. shows a prostate adenocarcinoma sample.

[0043]FIG. 4B shows immunohistochemistry of ovarian tissues.

[0044]FIG. 4C shows immunohistochemistry of cervical tissues.

[0045]FIG. 4D shows immunohistochemistry of lung tissues.

[0046]FIG. 4E shows summary of immunohistochemistry results of varioustissues.

[0047]FIG. 5A shows the growth curve of three ECV 304 cell lines.

[0048]FIG. 5B shows cell morphology of NIH/3T3 cells:

[0049] Normal wild type cells, cells overexpressing Ki-ras oncogene,cells overexpressing the LPAAT-β cDNA from a retroviral vector, and thelater from which the exogenous LPAAT-β gene has been removed by crerecombinase.

[0050]FIG. 5C shows the proliferation in low serum (2%) of 2 populationsof LPAAT-β over-expressing cells and subclones of those same populationsfrom which the exogenous LPAAT-β has been removed by cre recombinase.Also shown are normal, untransduced NIH/3T3 cells

[0051]FIG. 5D compares the proliferation in low serum (2%) ofpopulations of LNCAP cells transduced with either LPAAT-β or controlvectors.

[0052]FIG. 5E shows 6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamineat >20 μM is effective in blocking the proliferation of MCF-7 cells.

[0053]FIG. 6A compares tumor formation of LPAAT-β over-expressing cloneand control cells in nude mice.

[0054]FIG. 6B shows effect of6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine on growth ofNIH3T3-Ki-ras tumors in nude mice.

[0055]FIG. 6C shows effect of6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine on growth of B16murine melanoma cells in C57B/6 mice.

[0056]FIG. 6D shows effect of6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine on growth of murineLewis Lung Tumor cells in C57B/6 mice.

[0057]FIG. 6E shows effect of6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine on growth of humanDU145 Prostate Tumor Xenograft in nude mice.

[0058]FIG. 7 shows an example of the colorimetric assay.

[0059]FIG. 8 shows an example of the results obtained from assaying aplate of various compounds at 16 μM.

[0060]FIG. 9 shows concentration-dependent inhibition of LPAAT-β by6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] I. LPAAT-α and LPAAT-β: An Overview.

[0062] Northern blot analysis shows that LPAAT-α is expressed in allhuman tissues tested with the highest expression level found in skeletalmuscle (West et al. DNA Cell Biol. 16:691 (1997)). The uniformity ofLPAAT-α expression has also been found in additional tissues such asprostate, testis, ovary, small intestine, and colon (Stamps et al.,Biochem. J. 326: 455 (1997)) as well as in mouse tissues (Kume et al,Biochem. Biophys. Res. Commun. 237: 663 (1997)). A 2 kb and a 1.3 kbforms, possibly due to alternative utilization of polyadenylationsignals at the 3′-UTR, have been found in murine LPAAT-α mRNA (Kume etal., Biochem. Biophys. Res. Commun 237: 663 (1997)), whereas only onemajor human LPAAT-α mRNA of 2 kb in size has been detected by Northernanalysis. West et al., DNA Cell Biol. 16: 691 (1997); Stamps et al.,Biochem. J. 326: 455 (1997).

[0063] In contrast, LPAAT-β demonstrates a distinct tissue distributionof mRNA expression. West et al., DNA Cell Biol. 16: 691 (1997). LPAAT-βis most highly expressed in liver and heart tissues. LPAAT-β is alsoexpressed at moderate levels in pancreas, lung, skeletal muscle, kidney,spleen, and bone marrow; and at low levels in thymus, brain andplacenta. This differential pattern of LPAAT-β expression has beenconfirmed independently (Eberhardt et al., J. Biol. Chem. 272: 20299(1997)) with the only discrepancy being that high level, instead ofmoderate level, of LPAAT-β has been detected in pancreas, possibly dueto slight lot variations in commercial RNA blots (Clontech, Palo Alto,Calif.). In addition, moderate LPAAT-β expression has been found inprostate, testis, ovary, small intestine, and colon with the smallintestine containing relatively higher amounts. Eberhardt et al., J BiolChem 272: 20299 (1997). Within various brain sections, high expressionhas been found in the subthalamic nucleus and spinal cord; and least inthe cerebellum, caudate nucleus, corpus callosum, and hippocampus.LPAAT-β can also be detected in myeloid cell lines THP-1, HL-60, andU937 with the mRNA levels remaining the same with or withoutphorbal-ester treatment. The size difference between human LPAAT-α andLPAAT-β mRNA is consistent with the sequence data, in which LPAAT-α hasa longer 3′-UTR. The differential tissue expression pattern LPAAT-α andLPAAT-β mRNA would suggest these two genes are regulated differently andare likely to have independent functions. Therefore, a desirable featurein compounds that inhibit LPAAT activity is that they are specific ininhibiting one isoform of the enzyme over the other (i.e., LPAAT-β overLPAAT-α).

[0064] II. LPAAT-β and Cancer.

[0065] PA has been implicated in mitogenesis of several cell lines.English, Cell Signal 8: 341 (1996). PA level has been found to beincreased in either ras or fps transformed cell lines compared to theparental Rat2 fibroblast cell line (Martin et al., Oncogene 14: 1571(1997). To test whether LPAAT expression may be enhanced in certaintumor cells, the expression of LPAAT-α and LPAAT-β mRNA in human tumorpanel blots (Invitrogen, Carlsbab, Calif.) that contained tumor RNAs,isolated from various malignant tissues and RNAs from the normal tissuesin the surgical margins, were examined. Leung et al., DNA Cell Biol. 17:377 (1998). The same blots were also reprobed using cDNAs encodingphosphatidic acid phosphatase isoform PAP2-α; an enzyme that degrades,rather than generates, PA. Of a total of eight different tissuesexamined, LPAAT-β mRNA was found to be elevated in three tumors tissues(uterus, fallopian tube, and ovary), as compared to its expression inthe corresponding normal tissues. However, no significant difference wasfound in LPAAT-α mRNA level between the various tumor tissues and thenormal adjacent tissues. In two of the tumor tissues (fallopian tube andovary) where LPAAT-α mRNA was elevated, PAP2-α mRNA expression was foundto be suppressed, as it was also in tumors of the colon, rectum, andbreast.

[0066] Since the finding of differential expression of LPAAT-β mRNA incertain tumor versus normal tissues is based on Northern analysis of asingle specimen from a given tissue, more studies will be needed todetermine whether the relative elevation of LPAAT-β expression inselected tumor tissues can be applied and extended to similar tissuesderived from a larger number of donors. Leung et al., DNA Cell Biol. 17:377 (1998). Accordingly, in situ hybridization was used to compareLPAAT-β mRNA levels in breast, ovary, and prostate tumor samplesobtained from multiple independent donors (LifeSpan Biosciences,Seattle, Wash.). Specifically, the coding region of human LPAAT-β wasamplified by PCR from the plasmid pCE9. LPAAT-β with primers5′-GCATGAATTC AAAGGCCTAC GTCGACATGG AGCTGTGGCC GTG-3′ and 5′-GTCGACTCTAGACTACTGGG CCGGCTGCAC-3′. The resultant 870 bp PCR product was then cutwith EcoR I and XbaI for insertion in between the EcoR I and XbaI sitesof the in vitro transcription vector pDP18-T7/T3 (Ambion, Austin, Tex.)to generate the plasmid pDP_lptB. Serial tissue sections from paraffinarchival samples were hybridized with digoxigenin labeled riboprobestranscribed from either a T3 (sense) or T7 (antisense) transcriptioninitiation site present in the plasmid pDP_lptB linearized with eitherEcoR I (antisense) or Xba I (sense). The tissue sections from paraffinblocks were digested with proteinase K (20 μg/ml) for 4 minutes, thenhybridized with the antisense probe (1 μg/ml) at 60° C. for 22 hours andsubsequently washed with 2×SSC and 0.1×SSC at 50° C. The hybridizationsignals were detected with NBT/BCIP substrates using three cycles of analkaline phosphatase TSA amplification system (NEN Life Sciences,Boston, Mass.). The specimens were then counterstained with methylgreen. The signal was developed within 30 minutes at room temperature.The slides were then imaged using a digital camera mounted onto amicroscope.

[0067] Breast and ovary tissues were chosen for further in situhybridization study, as initial Northern analysis showed elevation ofLPAAT-β mRNA levels in tumors derived from the female reproductivetract. Prostate tissue was chosen, as it responds to steroid hormonesand contains ductal structures in a manner similar to breast and ovarytissues. Using an anti-sense cDNA probe, it was demonstrated thatexpression of the β isoform of this enzyme (LPAAT-β) was augmented inhuman tumor tissue in 10/11 ovarian, 14/20 breast, and 7/16 prostatebiopsies as compared to normal adjacent tissues. FIG. 1 shows an exampleof the results on a breast intraductal adenocarcinoma sample where thereis moderate increase in LPAAT-β mRNA level in the tumor samples (top 2panels) as evidenced by more dark-purple to brown spots compared toadjacent hyperplasia (bottom-left panel) and normal tissue (bottom-rightpanel). The slight increase in LPAAT-β mRNA staining in the hyperplasiasample (bottom-left panel) versus the normal sample (bottom-right panel)suggests that elevation occurs at an early stage of oncogenesis. FIG. 2shows an example of the results on another breast intraductaladenocarcinoma sample where there is large increase in LPAAT-β mRNAlevel in the tumor sample (left panel) as evidenced by more dark-purplespots versus the adjacent normal tissue (right panel). FIG. 3 showsthree examples of ovarian cancer samples where the LPAAT-β mRNA levelsare elevated and one example with undetectable level of LPAAT-β mRNA(lower right panel). FIG. 4A shows an example of the results on aprostate adenocarcinoma sample where there is moderate increase inLPAAT-β mRNA level in the tumor samples (left panel) as evidenced bymore dark-purple spots versus the adjacent normal tissue (right panel).In no cases have elevated levels of LPAAT-β mRNA expression been foundin the adjacent normal region from the same donor even in those cases ofbreast, ovarian, or prostate tumor where LPAAT-β mRNA levels happen tobe low or undetectable. The augmented expression of LPAAT-β in a highpercentage of tumor samples from breast (70%), ovary (91%), and prostatetissues (44%) would suggest that LPAAT-β overexpression may be acontributing factor for the development of these tumors.

[0068] To determine if increased transcription of LPAAT-β mRNA inselected tumor tissues can be extended to increased LPAAT-β proteinexpression in a wider range of tissues, a monoclonal antibody specificfor human LPAAT-β protein (MoAb 4B12) was generated based on the petidesequence, DLGERMVRENLKVW, derived from amino acids 155-168 of LPAAT-βprotein (BAbCO, Berkeley, Calif.). FIG. 4B shows an example of theresults on immunohistochemical staining (PhenoPath, Seattle, Wash.) withMoAb 4B12 at 1:4000 dilution of ovarian tissue where there issubstantial increase in LPAAT-β protein expression in the tumor samples(right panels) as evidenced by more intense brown stainings versus thenormal tissue (left panel). FIG. 4C shows an example of the results onimmunohistochemical staining (PhenoPath, Seattle, Wash.) with MoAb 4B12at 1:4000 dilution of cervical tissue where there is substantialincrease in LPAAT-β protein expression in the tumor samples (rightpanels) as evidenced by more intense brown stainings versus the normaltissue (left panel). There is also more staining in the surroundingstromal cells (indicated by arrows) in the tumor tissue vs the normaltissue, suggesting that the tumor may also induce LPAAT-β proteinexpression in the surrounding cells. FIG. 4D shows another example ofthe results on immunohistochemical staining (PhenoPath, Seattle, Wash.)with MoAb 4B12 at 1:4000 dilution of lung tissue where there isextensive increase in LPAAT-β protein expression in the tumor samples(right panels) as evidenced by more intense brown stainings versus thenormal tissue (left panel). FIG. 4E shows the summary ofimmnohistochemistry (IHC) results of the various tissue samples stainedby MoAb 4B12. The augmented expression of LPAAT-β in a high percentageof tumor samples again suggest that LPAAT-β overexpression may be acontributing factor for the development of these tumors and that LPAAT-βmay be a useful target for the development of anti-cancer compounds.

[0069] The aforementioned antibody may also be used for diagnostic andprognostic purposes when a tumor is present both on biopsies and inserum or plasma. For example, ELISA may be performed on serum to detectlung or ovarian cancer. It should be mentioned that currently there areno useful early diagnostics for these types of cancers.

[0070] The overexpression of LPAAT-β in selected tumor tissues wouldalso suggest the LPAAT-β protein may constitute a useful antigen for thedevelopment of tumor vaccines against those tumors where LPAAT-β isoverexpressed. Fong et al., Annu. Rev. Immunol. 18: 245 (2000);Schreurs, et al., Crit. Rev. Oncol. 11: 1 (2000). One such approach mayuse autologous dendritic cells, a type of potent antigen-presentingcells, to present LPAAT-β as a tumor-associated antigens for thegeneration of tumor-specific immunity through the MHC class I and IIprocessing pathways. Administration of dendritic cells loaded ex vivowith LPAAT-β as a therapeutic vaccine to patients with tumors withaugmented LPAAT-β expression may induce T cell-mediated tumordestruction.

[0071] To assess whether LPAAT-β overexpression in cells would lead tocertain phenotypic changes that are commonly observed in transformedcells, the growth and adherence characteristics of ECV304 cells(American Type Culture Collection, Richmond, Va.) expressing LPAAT-β(LPTb), expressing a catalytically inactive form of LPAAT-β (b-M8)whereby the arginine at position 175 was changed to alanine using theGeneEditor™ in vitro site-directed mutagenesis system (Promega, Madison,Wis.), or expressing green fluorescent protein (GFP) as a control werecompared. The aforementioned cells that express GFP may be considered tobe a non-limiting example of a “predetermined control,” according to thepreferred embodiments of the present invention. That is, such cells maybe used to gauge whether a cell is over- or under-expressing LPAAT-βDNA, RNA or protein. FIG. 5A shows the growth curve of these three celllines. Each cell line was seeded at 200,000 cells per 60 mm plate. Thecell numbers at various times after seeding were determined by countingwith a hemacytometer. The growth rate of the three cell lines weresimilar until they reached confluence at 100 hours after plating. Afterconfluence, the LPTb cells were able to continue to proliferate, whilethe b-M8 and GFP cells' growth started to level off. This demonstratedthat ECV304 cells overexpressing LPAAT-β could continue to grow andcould form a plurality of layers after they had formed a confluentmonolayer of cells. The proliferation of the cells with the inactivemutant or the control cells slowed down after confluence. The loss ofcontact inhibition and the propensity for growth to an unusually highcell density are changes commonly observed in tumorigenesis. The factthat the inactive LPAAT-β mutant (b-M8) expressing cells, like thevector control cells, are constrained by density-dependent inhibition ofcell division strongly suggests that the capacity to overcome contactinhibition may be due to increases in LPAAT-β enzymatic activity. Thedevelopment of compounds that inhibit LPAAT-β enzymatic activity mayreverse the growth pattern and hence tumorigenesis in cells withabnormally high level of LPAAT-β expression.

[0072] To determine if the observation from LPAAT-β expressing ECV304cells can be extended to other cell types and to animal models oftumorigenesis, LPAAT-β cDNA was inserted into a retroviral expressionvector, pLOXSN, for the generation of recombinant viral stocks in apackaging cell line, PT67 (Clontech, Palo Alto, Calif.), fortransduction into various cell lines. The vector pLOXSN was derived frompLXSN with insertion of a 19 bp oligonucleotide coding for the locus ofrecombination (lox) signal sequence as well as a ClaI recognition siteinto the NheI site within the 3′-LTR region of pLXSN. Miller and RosmanBioTechniques 7: 980 (1989); Hoess. and Abremski, Nucleic Acid and Mol.Biol. 4: 99 (1990). This lox sequence will be duplicated within the5′-LTR region during viral replication. Hence the sequence in betweenthe two lox sites located within the 5′- and the 3′-LTR can be excisedif required in the presence of the enzyme cre recombinase supplied intrans from a separate retroviral vector with a different selectablemarker.

[0073] Over-expression of the normal cellular LPAAT-β cDNA in NIH/3T3cells was associated with transformation in 3 out of 9 transducedpopulations. As is the case with normal cellular proto-oncogenes,over-expression of LPAAT-β is not sufficient, but may contribute totransformation along with other, spontaneous events. FIG. 5B showsexamples of cell morphology of NIH/3T3 cells: a bulk populationtransfected with a plasmid overexpressing the Ki-ras oncogene (top leftpanel), a selected clone transduced with a retroviral vectoroverexpressing LPAAT-β (Hc2; lower left panel) and cells with theLPAAT-β cDNA excised using the lox-cre recombination in the lower leftand normal, untransduced cells (top right panel). Sauer, Methods 14: 381(1998). The control untransduced cells exhibited normal fibroblastmorphology and grew as a contact-inhibited, adherent monolayer (topright panel). In contrast, both the Ki-ras and LPAAT-β overexpressingcells were more elongated and spiked, were not contact-inhibited andformed foci typical of transformation of these immortalized fibroblasts.After removal LPAAT-β transgene by lox-cre recombination from the Hc2clone (bottom right panel), this transformed morphology was lost,suggesting that LPAAT-β overexpression is a contributing factor to thistransformation phenotype rather than being the result entirely ofspontaneous events during in vitro passage.

[0074] Another common parameter of cancer cells is a reduced requirementfor elements present in serum. FIG. 5C compares the growth profiles oftransduced populations of NIH/3T3 cells in low (2%) serum. Twoindependent populations (LPT Hc2, LPT L bulk) overexpressing LPAAT-βhave an increased ability to proliferate compared to a control vectorclone expressing alkaline phosphatase (APc1) and those correspondingpopulations with deletion of the LPAAT-β transgene by lox-crerecombination (LPT Hc2cre, LPT L bulkcre), suggesting that LPAAT-βoverexpression is a contributing factor to this transformed phenotype ofproliferation with a reduced requirement for growth factors.

[0075] Similarly, out of a total of 12 populations of human prostateLNCaP cells (American Type Culture Collection, Manassas, Va.) transducedwith LPAAT-β expressing vector, most of them show augmentedproliferation in low serum when compared to control cells (FIG. 5D).

[0076] To determine whether administration of LPAAT-β inhibitor wouldhave any effect on cell proliferation in tissue culture, proliferationof human breast tumor MCF-7 cells in microplates were measured byCyQUANT analysis using a green-fluorescent nucleic acid stain optimizedto produce a linear detection range from 50 to 50,000 cells in 200 μlvolume (Molecular Probes, Eugene, Oreg.) in the presence of variousconcentrations of a LPAAT-β inhibitor. FIG. 5E shows the triazinecompound shows 6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine at ≧20μM is effective in blocking the proliferation of MCF-7 cells.

[0077] To determine if LPAAT-β overexpression would contribute totumorigenesis in mice, 2×10⁶ NIH3T3 cells overexpressing LPAAT-β (LPAATvector) and control cells were injected subcutaneously into nude mice.FIG. 6A shows tumor could be detected after 14 days from the LPAAT-βoverexpressing cells, while no tumor formation was detected in vectorcontrol cells after 28 days. The cells with the transgene removed bylox-cre recombination showed delay of tumor formation compared toLPAAT-β overexpressing cells by ˜7days. Recovery and analysis of thelox-cre cells from mice showed that there had been in vivo selection ofa small sub-population that had not been recombined to remove theLPAAT-β transgene. This analysis demonstrated that the only cells toform tumors retained the original LPAAT vector and indeed had a highlevel of LPAAT activity as well as G418 resistance (the neo gene is alsoremoved along with LPAAT-β during the cre-lox procedure). These datashow LPAAT-β overexpression is a contributing factor for tumorigenesisin vivo.

[0078] To determine whether administration of LPAAT-β inhibitor wouldhave any effect on tumor growth in mice, 5×10⁵ NIH/3T3 cellsoverexpressing the oncogene Ki-ras were injected subcutaneously intonude mice. An LPAAT-β inhibitor6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine, at concentrationsthat range from 10 mg/Kg to 100 mg/Kg of mouse body weight was injectedintra-peritoneally on day 1, 2, 3 and 4 after injection of tumor cells.The size of tumors was then measured on day 8. FIG. 6B shows the volumeof the tumors in mice is decreased as the concentration of the LPAAT-βinhibitor increases, suggesting that administration of this LPAAT-βinhibitor is efficacious in slowing down tumor growth in vivo.

[0079] In addition to slowing down tumor growth of NIH3T3/Ki-ras cellsin nude mice, 6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine hasalso been found to decrease the growth of B16 melanoma cells (FIG. 6C)and Lewis Lung tumor cells (FIG. 6D) in syngeneic mice as well as thegrowth of human DU145 prostate tumor cells in nude mice in a xenograftstudy (FIG. 6E).

[0080] Analysis and characterization of phospholipids and other complexlipids represent another strategy to measure effects of small moleculeinhibitors on phospholipid metabolizing enzymes involved in tumorprogression, including but not limited to, LPAAT-β. Measurements ofphospholipids and other complex lipids may be derived from cell linescultured in vitro, from tissue or plasma in vivo (e.g., murine or otheranimal studies), or from human subjects (e.g., phlebotomy or biopsy).Phospholipids, which are the primary constituents of a cellular bilayer,contain a universal phosphoric acid residue connected to a glycerolbackbone. Phospholipid classes are defined by the chemical identity ofthe “head group” on the phosphoric acid moiety. However, eachphospholipid class is often a complex mixture of discrete molecularspecies due to the fact that the glycerol backbone has two substituentsresiding at the Sn1 and Sn2 position of attachment. The substituents areacyl chains and typically consist of long chain fatty acids but may alsoinclude a long chain ether, acetyl, or hydroxyl group. Chemicalmeasurements of phospholipids can involve a variety of analyticalmethods including, but not limited to, HPLC-MS (High Performance LiquidChromatography-Mass Spectrometry), HPLC-MS/MS (High Performance LiquidChromatography-Tandem Mass Spectrometry), one or two dimensional TLC(Thin Layer Chromatography), and radiometry. While all the statedmethods can be used to quantitate bulk mass changes in a particularphospholipid class of interest, mass spectrometry offers the uniqueability to measure all molecular species within a phospholipid class ina single measurement with a high degree of precision.

[0081] The above approach is demonstrated by performing HPLC-MS analysesof phospholipid extracts from murine NIH/3T3 immortalized fibroblasts,both normal wild type, βHc2 cells (i.e., overexpressing LPAAT-β, andHc2cre cells (i.e., LPAAT-β gene removed by site-specificrecombination). Analysis of phosphatidylinositol in these cellpopulations clearly indicate a combined effect of LPAAT-β overexpressionand cellular transformation for the Hc2 population over that of thenormal wild type. This effect is characterized by an increase inunsaturated (i.e., palmitate and stearate) and monounsaturated (i.e.,oleate) fatty acyl chains indicated by an increased molecular abundanceof ions at m/z 807, 833, 835, 861, and 863 which correspond most likelyto phosphatidylinositol species with acyl chains designated as16:0-16:1, 16:1-18:1 (and/or 16:0-18:2), 16:0-18:1, 18:1-18:1 (and/or18:0-18:2), and 18:0-18:1, respectively. While multiple molecularspecies may reside at the same nominal mass, these species can bedifferentiated by tandem (MS/MS) mass spectrometry methods.Additionally, note that actual determination of positional location(i.e., Sn1 versus Sn2) requires other analytical methods and only themost prevalent configuration is listed here. In addition to the increasein unsaturated and monounsaturated acyl chains in the LPAAT-βoverexpressing population (βHc2), there is also a corresponding decreasein polyunsaturated (i.e., arachidonate) fatty acyl chains at m/z 857(16:0-20:4) and m/z 885 (18:0-20:4). Removal of the LPAAT-β transgeneresults in phosphatidylinositol distributions similar to that of thenormal wild type 3T3 cells.

[0082] In summary, endogenous LPAAT-β expression is detected at highlevels by both in situ hybridization and immunohistochemistry inparticular tumor tissues and often in surrounding stroma and isassociated with tumor progression. LPAAT-β overexpression appears tocontribute reversibly to transformation and tumorigenesis ofimmortalized rodent cells and may also contribute to increasedtransformation of weakly tumorigenic human cell lines. Compoundsselected from screening of LPAAT-β inhibitors from different structuralfamilies can inhibit proliferation of numerous tumor cell lines invitro. Both nude and immunocompetent mice can tolerate at least 100mg/kg/day for 4-5 days of the6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine maintaining bodyweight and overall health with no discernable gross pathology. Thiscompound can inhibit the growth of numerous tumor models in mice and maybe a tumor-static compound.

[0083] III. LPAAT-β Inhibitors.

[0084] In one aspect, the compounds of the present invention relate totriazines of the Formula:

[0085] wherein,

[0086] R¹ is halo, hydroxy, alkylmercapto, mercapto, alkoxy, aryloxy orsubstituted amino;

[0087] R², R³, R⁴ and R⁵, each of which may be same or different, arehydrogen, alkyl, substituted alkyl, alkenyl, alkynyl, aryl orsubstituted aryl; or

[0088] R² and R³ or R⁴ and R⁵, together with the nitrogen to which theyare attached, form a piperidine, piperazine, or a morpholine ring; or

[0089] pharmaceutically acceptable salts thereof.

[0090] As used herein, “alkyl” refers to straight- or branched-chainhydrocarbons having from 1 to 10 carbon atoms and more preferably 1 to 8carbon atoms which includes, by way of example, methyl, ethyl, n-propyl,i-propyl, n-butyl, t-butyl and the like.

[0091] The term “alkyl” also refers to an “unsaturated alkyl” moiety,which means that it contains at least one alkene or alkyne moiety.“Alkene” or “alkenyl” refers to a group consisting of at least twocarbon atoms and at least one carbon-carbon double bond. “Alkyne” or“alkynyl” refers to a group consisting of at least two carbon atoms andat least one carbon-carbon triple bond. The alkyl moiety, whethersaturated or unsaturated, may be branched, non-branched, or cyclic.

[0092] “Substituted alkyl” refers to an alkyl group, preferablycontaining from 1 to 10 carbon atoms, having from 1 to 5 substituentsincluding halogen, hydroxyl, alkyl, aryl or substituted amino. Apreferred substituted alkyl group is trifluromethyl.

[0093] “Alkoxy” refers to the group “alkyl—O—” which includes, by way ofexample, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, t-butoxy andthe like.

[0094] “Substituted amino” refers to the group —NRR, wherein each Rgroup is independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, or the Rgroups can be joined together with the nitrogen to form a heterocyclicring (e.g., piperidine, piperazine, or a morpholine ring).

[0095] “Aryl” refers to an unsaturated aromatic carbocyclic group of 6to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl).

[0096] “Substituted aryl” refers to aryl group which are substitutedwith 1 to 3 substituents selected from hydroxy, alkyl, substitutedalkyl, alkoxy, amino, aryl, —O—(CH₂)_(n)—O— (wherein n is an integerfrom 1 to 3), —(CH₂)_(m)— (wherein m is an integer from 3 to 5) orhalogen.

[0097] “Cycloalkyl” refers to cyclic alkyl groups containing between 3and 8 carbon atoms having a single cyclic ring including, by way ofexample, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like.

[0098] “Halogen” or “halo” refers to fluoro, chloro, bromo, iodo. Mostpreferred halogens are chloro and fluoro.

[0099] “Mercapto” refers to the group —SR wherein the R group isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, aryl or substituted aryl. The term“alkylmerecapto” refers to the group —SR when R is alkyl, substitutedalkyl or cycloalkyl.

[0100] Compounds of the preferred embodiments of the present inventioninclude those compounds in Table 1. LPAATβ CT Colorimetric Assay NumberStructure IC₅₀ (nM) 31867

750 31942

400 31978

1,000 32028

200 32042

200 32099

650 116988

160 117147

3,800 31888

3,100

[0101] Preferred compounds include, but are not limited to,6-chloro-N-(4-methoxy-phenyl)-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,N-butyl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-isopropyl-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,N-tert-butyl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine,(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-naphthalen-1-yl-amine,N-tert-butyl-6-chloro-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-cyclohexyl-N′-isopropyl-[1,3,5]triazine-2,4-diamine,2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-2-methyl-propan-1-ol,6-chloro-N-isopropyl-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chloro-phenyl)-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine,N-allyl-6-chloro-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine,2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-ethanol,N-tert-butyl-6-chloro -cyclopentyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-methoxyphenyl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-indan-5-yl-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chloro-phenyl)-N′-propyl-[1,3,5]triazine-2,4-diamine,N-(4-chloro-phenyl)-6-methoxy-N′-propyl-[1,3,5]triazine-2,4-diamine andN-(4-chloro-phenyl)-6-methylsulfanyl-N′-phenyl-[1,3,5]triazine-2,4-diamine.

[0102] Most preferred compounds include, but are not limited to,6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine,N-tert-butyl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chlorophenyl)-N′-(4-methoxyphenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chlorophenyl)-N′-phenyl-[1,3,5]-triazine-2,4-diamine.

[0103] The compounds of the preferred embodiments of the presentinvention inhibit LPAAT-β and thereby inhibit cell proliferation.Therefore, the compounds of the preferred embodiments of the presentinvention may be useful in the treatment of cancer. The types of cancerthat may be treated with the compounds of the preferred embodiments ofthe present invention include, but are not limited to, prostate, breast,lung, ovarian, brain, cervical, colon or bladder cancer, and not limitedto tumor cells expressing high levels of LPAAT-β as evidenced by thedecrease in NIH/3T3 Ki-ras tumor cell growth in vitro and in vivo whentreated with, 6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine.

[0104] IV. Pharmacological Compositions, Therapeutic and OtherApplications.

[0105] The compound of the present invention, or its pharmaceuticallyacceptable salt, can be administered to a human patient per se, or inpharmacological compositions where it is mixed with pharmaceuticallyacceptable carriers or excipient(s). Techniques for formulation andadministration of drugs may be found in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition.

[0106] A. Routes of Administration.

[0107] Suitable routes of administration may include, withoutlimitation, oral, rectal, transmucosal or intestinal administration orintramuscular, subcutaneous, intramedullary, intrathecal, directintraventricular, intravenous, intraperitoneal or intranasal injections.

[0108] Alternately, one may administer the compound in a local ratherthan systemic manner, for example, via injection of the compounddirectly into a solid tumor, often in a depot or sustained releaseformulation.

[0109] Furthermore, one may administer the drug in a targeted drugdelivery system, for example, in a liposome coated with tumor-specificantibody. The liposomes will be targeted to and taken up selectively bythe tumor.

[0110] B. Composition/Formulation.

[0111] Pharmacological compositions of the compounds and thepharmaceutically acceptable salts thereof are preferred embodiments ofthis invention. Pharmacological compositions of the present inventionmay be manufactured by processes well known in the art; e.g., by meansof conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses.

[0112] Pharmacological compositions for use in accordance with thepresent invention thus may be formulated in a conventional manner usingone or more pharmaceutically acceptable carriers comprising excipientsand auxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

[0113] For injection, the compounds of the invention may be formulatedas sterile aqueous solutions, preferably in physiologically compatiblebuffers such as Hanks' solution, Ringer's solution, or physiologicalsaline buffer. For transmucosal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0114] For oral administration, the compounds can be formulated readilyby combining the active compounds with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmacological preparations fororal use can be made with the use of a solid excipient, optionallygrinding the resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are, in particular, fillers such assugars, including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

[0115] Dragee cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

[0116] Pharmacological compositions which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with a fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

[0117] For buccal administration, the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0118] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0119] The compounds may be formulated for parenteral administration,e.g., by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, e.g., in ampules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

[0120] Pharmacological compositions for parenteral administrationinclude sterile aqueous solutions of the active compounds in watersoluble form. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

[0121] Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0122] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0123] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation (see, forexample, U.S. Pat. No. 5,702,717 for a biodegradable depot for thedelivery of a drug). Such long acting formulations may be administeredby implantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compounds may beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.The pharmacological compositions herein also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

[0124] The compounds of the invention that inhibit LPAAT-β may beprovided as physiologically acceptable salts wherein the claimedcompound may form the negatively or the positively charged species.Examples of salts in which the compound forms the positively chargedmoiety include, without limitation, quaternary ammonium (definedelsewhere herein), salts such as the hydrochloride, sulfate, carbonate,lactate, tartrate, maleate, succinate, etc. formed by the reaction of anamino group with the appropriate acid.

[0125] C. Dosage.

[0126] Pharmacological compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve its intended purpose.

[0127] More specifically, a therapeutically effective amount means anamount of compound effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated.

[0128] Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art, especially in lightof the detailed disclosure provided herein.

[0129] For any compound used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromcell culture assays. For example, a dose can be formulated in animalmodels to achieve a circulating concentration range that includes theIC₅₀ as determined in cell culture (i.e., the concentration of the testcompound which achieves a half-maximal inhibition of LPAAT-β activity).Such information can be used to more accurately determine useful dosesin humans.

[0130] Toxicity and therapeutic efficacy of the compounds describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals for determining the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio between LD₅₀ and ED₅₀. Compounds which exhibit hightherapeutic indices are preferred. The data obtained from these cellculture assays and animal studies can be used in formulating a range ofdosage for use in human. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (seee.g., Fingl, et al., in “The Pharmacological Basis of Therapeutics,”(1975), Chapter 1, pp. 1).

[0131] Dosage amount and interval may be adjusted individually toprovide plasma levels of the active moiety which are sufficient tomaintain LPAAT-β inhibitory effects, or minimal effective concentration(MEC). The MEC will vary for each compound but can be estimated from invitro data; e.g., the concentration necessary to achieve 50-90%inhibition of LPAAT-β using the assays described herein. Dosagesnecessary to achieve the MEC will depend on individual characteristicsand route of administration. However, HPLC assays or bioassays can beused to determine plasma concentrations.

[0132] Dosage intervals can also be determined using MEC value.Compounds should be administered using a regimen which maintains plasmalevels above the MEC for 10-90% of the time, preferably between 30-90%and most preferably between 50-90%.

[0133] In cases of local administration or selective uptake, theeffective local concentration of the drug may not be related to plasmaconcentration.

[0134] The amount of composition administered will, of course, bedependent on the subject being treated, on the subject's weight, theseverity of the affliction, the manner of administration and thejudgment of the prescribing physician. An exemplary systemic dailydosage is about 5 to about 200 mg/kg of body weight. Normally, fromabout 10 to about 100 mg/kg of body weight of the compounds of thepreferred embodiments of the present invention, in one or more dosagesper day, is effective to obtain the desired results. One of ordinaryskill in the art can determine the optimal dosages and concentrations ofthe compounds of the preferred embodiments of the present invention withonly routine experimentation.

[0135] The compounds of the preferred embodiments of the presentinvention are substantially pure and preferably sterile. The phrase“substantially pure” encompasses compounds created by chemical synthesisand/or compounds substantially free of chemicals which may accompany thecompounds in the natural state, as evidenced by thin layerchromatography (TLC) or high performance liquid chromatography (HPLC).

[0136] D. Other Applications

[0137] The compounds of the preferred embodiments of the presentinvention may be employed not only for therapeutic purposes, but also asaids in performing research in vitro. For example, the compounds of thepreferred embodiments of the present invention may be used to studybiochemical pathways that would require the inhibition of LPAAT-β toelevated levels of LPA. Inhibition of LPAAT-β may result in theprolonged or limited activity of biochemical pathways that depend on, orrespond to, elevated levels of LPA.

[0138] Additionally, a cell culture medium comprising the compounds ofthe preferred embodiments of the present invention is within the scopeof the invention.

[0139] V. Assays for LPAAT-β DNA, RNA and Protein.

[0140] DNA molecules encoding the human LPAAT-β gene, or fragmentsthereof, can be used to detect the level of LPAAT-β gene expression intissue samples. Such a detection method can be used, for example, tocompare the amount of LPAAT-β RNA in a sample obtained from normaltissue and in a sample isolated from methotrexate-resistant tumortissue. The presence of relatively low levels of LPAAT-β RNA in thetumor sample would indicate that methotrexate resistance is due, atleast in part, to underexpression of the LPAAT-β gene.

[0141] In testing a tissue sample for LPAAT-β RNA using a nucleic acidhybridization assay, RNA can be isolated from tissue by sectioning on acryostat and lysing the sections with a detergent such as SDS and achelating agent such as EDTA, optionally with overnight digestion withproteinase K. Such tissue may be obtained by biopsy. A preferredquantity of tissue is in the range of 10-100 milligrams. Protein may beremoved by phenol and chloroform extractions, and nucleic acids areprecipitated with ethanol. RNA may be isolated by chromatography on anoligo dT column and then eluted from the column. Further fractionationcan also be carried out according to methods well known to those ofordinary skill in the art.

[0142] A number of techniques for molecular hybridization are used forthe detection of DNA or RNA sequences in tissues. When large amounts oftissue are available, analysis of hybridization kinetics provides theopportunity to accurately quantitate the amount of DNA or RNA present,as well as to distinguish sequences that are closely related but notidentical to the probe. Reactions are run under conditions ofhybridization (T_(m)−25° C.) in which the rate of re-association of theprobe is optimal. Wetmur et al., J. Mol. Biol. 31:349 (1968). Thekinetics of the reaction are second order when the sequences in thetissue are identical to those of the probe; however, the reactionexhibits complex kinetics when probe sequences have partial homology tothose in the tissue. Sharp et al., J. Mol. Biol. 86:709 (1974).

[0143] The concentration of probe to cellular RNA is determined by thesensitivity desired. To detect one transcript per cell would requireabout 100 pg of probe per mg of total cellular DNA or RNA. The nucleicacids are mixed, denatured, brought to the appropriate saltconcentration and temperature, and allowed to hybridize for variousperiods of time. The rate of reassociation can be determined byquantitating the amount of probe hybridized either by hydroxyapatitechromatography (Britten et al., Science 161:529 (1968)) or by S1nuclease digestion (Sutton, Biochim. Biophys. Acta 240:522 (1971)).

[0144] Another method of hybridization is the Northern Blot technique.The particular hybridization technique is not essential to theinvention, and any technique commonly used in the art is within thescope of the present invention. Typical probe technology is described inU.S. Pat. No. 4,358,535, incorporated by reference herein. For example,hybridization can be carried out in a solution containing 6×SSC (10×SSC:1.5 M sodium chloride, 0.15 M sodium citrate, pH 7.0), 5×Denhardt's(1×Denhardt's: 0.2% bovine serum albumin, 0.2% polyvinylpyrrolidone,0.02% Ficoll 400), 10 mM EDTA, 0.5% SDS and about 10⁷ cpm ofnick-translated DNA for 16 hours at 65° C.

[0145] The aforementioned hybridization assays are particularly wellsuited for preparation and commercialization in kit form, the kitcomprising a carrier means compartmentalized to receive one or morecontainer means (vial, test tube, etc.) in close confinement, with eachcontainer means comprising one of the separate elements to be used inhybridization assay. For example, there may be a container meanscontaining LPAAT-β DNA molecules suitable for labeling by “nicktranslation,” or containing labeled LPAAT-β DNA or labeled LPAAT-β RNAmolecules. Further container means may contain standard solutions fornick translation of DNA comprising DNA polymerase I/DNase I andunlabeled deoxyribonucleotides.

[0146] Antibodies to human LPAAT-β protein can be obtained using theproduct of an LPAAT-β expression vector as an antigen. The preparationof polyclonal antibodies is well-known to those of skill in the art.See, for example, Green et al., “Production of Polyclonal Antisera,” inImmunochemical Protocols (Manson, ed.), pp. 1-5 (Humana Press 1992).Alternatively, an LPAAT-β antibody of the present invention may bederived from a rodent monoclonal antibody (MAb). Rodent monoclonalantibodies to specific antigens may be obtained by methods known tothose skilled in the art. See, for example, Kohler and Milstein, Nature256:495, 1975, and Coligan et al. (eds.), Current Protocols inImmunology, 1:2.5.1-2.6.7 (John Wiley & Sons 1991) [hereinafter“Coligan”]. Briefly, monoclonal antibodies can be obtained by injectingmice with a composition comprising an antigen, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to the antigen, culturing the clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

[0147] MAbs can be isolated and purified from hybridoma cultures by avariety of techniques that are well known in the art. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in Methods inMolecular Biology, 10:79 (Humana Press, Inc. 1992). A LPAAT-β antibodymay also be derived from a subhuman primate antibody. General techniquesfor raising therapeutically useful antibodies in baboons may be found,for example, in Goldenberg et al., International Patent Publication No.WO 91/11465 (1991), and in Losman et al., Int. J. Cancer 46:310 (1990).

[0148] Alternatively, a therapeutically useful LPAAT-β antibody may bederived from a “humanized” monoclonal antibody. Humanized monoclonalantibodies are produced by transferring mouse complementary determiningregions from heavy and light variable chains of the mouse immunoglobulininto a human variable domain, and then, substituting human residues inthe framework regions of the murine counterparts. The use of antibodycomponents derived from humanized monoclonal antibodies obviatespotential problems associated with the immunogenicity of murine constantregions. General techniques for cloning murine immunoglobulin variabledomains are described, for example, by the publication of Orlandi etal., Proc. Nat'l. Acad. Sci. USA 86:3833 (1989). Techniques forproducing humanized MAbs are described, for example, by Jones et al.,Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988);Verhoeyen et al., Science 239:1534 (1988); Carter et al., Proc. Nat'lAcad. Sci. USA 89:4285 (1992); Sandhu, Crit. Rev. Biotech. 12: 437(1992); and Singer et al., J. Immun. 150:2844 (1993), each of which ishereby incorporated by reference.

[0149] As an alternative, a LPAAT-β antibody of the present inventionmay be derived from human antibody fragments isolated from acombinatorial immunoglobulin library. See, for example, Barbas et al.,METHODS: A Companion to Methods in Enzymology 2:119 (1991); and Winteret al., Ann. Rev. Immunol. 12:433 (1994) which are incorporated hereinby reference. Cloning and expression vectors that are useful forproducing a human immunoglobulin phage library can be obtained, forexample, from STRATAGENE Cloning Systems (La Jolla, Calif.). Inaddition, a LPAAT-β antibody of the present invention may be derivedfrom a human monoclonal antibody. Such antibodies are obtained fromtransgenic mice that have been “engineered” to produce specific humanantibodies in response to antigenic challenge. In this technique,elements of the human heavy and light chain locus are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the endogenous heavy chain and light chain loci.The transgenic mice can synthesize human antibodies specific for humanantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7:13 (1994); Lonberg etal., Nature 368:856 (1994); and Taylor et al., Int. Immun. 6:579 (1994).

[0150] Having now generally described this invention, the same will beunderstood by reference to the following examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLE 1 Production of Recombinant LPAAT-β for Various Assays

[0151] For the construction of Baculovirus expression vectors, thefull-length human LPAAT-β cDNA was amplified by PCR from the DNAtemplate pCE9.LPAAT-β (West et al., DNA Cell Biol. 16:691-701 (1997))using the primers 5′- TGATATCCGA AGAAGATCTT ATGGAGCTGT GGCCGTGTC-3′(o1pb1F) and 5′-CAGGCTCTAG ACTACTGGGC CGGCTGCAC-3′ (o1pb1R). The ˜870 bpfragment generated was reamplified by PCR using the primers 5′ CCTACGTCGACATGGAACA AAAATTGATA TCCGAAGAAG ATC-3′ (o1pb2F) and 5′-CAGGCTCTAGACTACTGGGC CGGCTGCAC-3′ (o1pb1R). The ˜890 bp fragment generated wasthen cleaved with Sal I and Xba I for insertion into pFastBac™ HTcvector (Life Technologies, Gaithersberg, Md.) between the Sal I and XbaI sites for the generation of the plasmid pFB.LPAAT-β. This plasmid wasthen transformed into E. coli DH10Bac™ (Life Technologies, Gaithersberg,Md.) for the generation of recombinant Bacmid DNA for transfection intoHighFive (Invitrogen, San Diego, Calif.) or SF9 insect cells for theproduction of recombinant Baculovirus stocks using the protocoldescribed in the Bac-to-Bac® Baculovirus Expression System (LifeTechnologies, Gaithersberg, Md.), a eukaryotic expression system forgenerating recombinant baculovirus through site- specific transpositionin E. coli. Viral stocks harvested from the transfected cells can thenbe used to infect fresh insect cells for the subsequent expression ofLPAAT-β fusion protein with a polyhistidine tag and a myc-epitope nearits N-terminus. The membrane fraction from these Sf9 cells would be thesource of LPAAT enzyme.

EXAMPLE 2 Preparation of Cell Membranes from Sf9 Cells

[0152] For the preparation of membranes From Sf9 Cells, all steps areperformed on ice or at 4° C. Sf9 cell pellets (˜10⁸ cells) were thawedand resuspended in 1-2 ml of buffer A (20 mM Hepes, pH 7.5, 1 mM DTT, 1mM EDTA, 20% w/v glycerol, 1 mM Benzamidine, 1 μg/ml soybean trypsininhibitor (SBTI), 1 μg/ml pepstatin A) w/o DTT but with 1 mM Pefabloc.The cells were lysed by sonication using a Branson Sonifier at output=2,duty cycle=2, 10 pulses each at 10s. with the tip of small sonicatorprobe submerged but not touching the walls. DTT was then added to 1 mMfrom a 1 M stock. The samples were centrifuged at 1500 rpm for 5 min.The low speed supernatant was saved and centrifuged (TLA 100.3 rotor,polycarbonate tubes, 2 ml/tube or 1.5 ml/tube minimum) at 100000×g for 1hr. The high speed pellet was resuspend in Buffer A with a probesonicator (10 pulses @ output #2 and duty cycle 20%) as a source ofLPAAT enzyme.

EXAMPLE 3 Assay of LPAAT-β Activity

[0153] LPAAT-β catalyzes the transfer of an acyl group from a donor suchas acyl-CoA to LPA. The transfer of the acyl group from acyl-CoA to LPAleads to the release of free CoA, which can be reacted with the thiolreagent, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). The reactionbetween DTNB and the free sulfhydryl group from CoA generates ayellow-colored product, 3-carboxylato-4-nitrothiophenolate (CNP), thatabsorbs at 413 nm. LPAAT-β derived from Sf9 cell membrane overexpressingLPAAT-β were resuspended in HEPES saline buffer (20 mM HEPES pH 7.5, 150mM NaCl), 1 mg/ml BSA and 72 μl aliquots were distributed into 96-wellmicrotiter plates. 8 μl of compound of interest at 200 μM dissolved in100% DMSO was added into each well. 20 μl of 1 mM 18:1-CoA and 1 mMsn-1-18:1 lysoPA was then added to each well to initiate the reactionand allowed to run at room temperature for 25 min. 100 μl of 1 mM DTNBin 100% ethanol was then added to each well to quench the reaction andfor color development. The absorbance at 405 nm, measured using aspectrophotometer plate reader, is proportional to the activity ofLPAAT-β in the sample. This calorimetric assay was used for the highthroughput screening of LPAAT inhibitors. Compounds that showed >50%inhibition of the change in absorbance at 405 nm compared to controlwere selected for a secondary assay. FIG. 7 shows an example of thecolorimetric assay of which the time course of color development isdependent on the amount of LPAAT enzyme added. FIG. 8 shows an exampleof the results obtained from assaying a plate of various compounds at 16μM. Compounds that gave a reading of less than 0.06 arbitrary units(indicated by arrow on right margin) were selected for further study.

[0154] A secondary assay for LPAAT activity in cell extracts based oneither the conversion of fluorescent NBD-LPA to NBD-PA (West, et al.,DNA Cell Biol. 6: 691-701, 1997) or [¹⁴C]LPA to [¹⁴C]PA using TLCanalysis was used to screen compounds that showed >50% inhibition ofLPAAT activity in the primary calorimetric assay. The radiometric assaywas carried out in Sf9 cell membrane overexpressing LPAAT-β resuspendedin HEPES-saline buffer, pH 7.5, 1 mg/ml BSA, 1 mM EDTA and 200 μM[¹⁴C]18:1-CoA and 200 μM sn-1-18:1 lysoPA. The samples were incubated 7min at 37° C., extracted into organic solvent (CHCl₃/CH₃OH/HCl at33/66/1), before loading onto TLC plates. A more detailed protocol forthe radiometric assay is described below:

[0155] Specifically, this LPAAT assay is a modification of theacyltransferase assay published previously (Hollenback and Glomset,Biochemistry 37: 363-376 (1999)).

[0156] 1. The basic assay, in a total vol of 50 μl, employs a solutionof substrates and the protein sample. Total assay volume, as well as thevolume of each solution, can be changed to fit an experiment. Inaddition, other compounds, ex inhibitors and activators, can be includedin the assay as well.

[0157] 2. To prepare the solution of substrates:

[0158] a. Stocks of Hepes (pH 7.5), NaCl, EDTA, BSA and acyl-CoA (fromSerdery or Sigma) are mixed with water to make the appropriateconcentration of each compound. This can be varied from assay-to-assay,but the final reaction mix is about 50 mM Hepes, 100 mM NaCl, 1 mM EDTA,1 mg/ml BSA and 0-400 μM acyl-CoA.

[0159] b. The lysoPA (from Avanti) is typically stored in chloroform andthe ¹⁴C-labeled acyl-CoA (from Amersham) is typically stored inwater/ethanol=1:1. Appropriate amounts of each solution are added the toa 12×75 mm borosilicate glass test tube and dry the solvent under N₂ orAr. An appropriate volume of the solution prepared in 2a is added to thelysoPA and ¹⁴C-labeled acyl-CoA. The lipids are resuspend by sonicationfor 15 sec in a bath sonicator. The resulting suspension is thenincubated (with occasional gentle vortexing) for about 10 minutes atroom temp. The sn-1-16:0 lysoPA may require brief warming of the solventto solubilize it. The concentration of lysoPA and ¹⁴C-labeled acyl-CoAcan vary, but typically the final lysoPA concentration ranges between 0and 400 μM and the ¹⁴C-labeled acyl-CoA specific activity ranges between0.5 and 2 Ci/mol.

[0160] 3. Protein sample: varies from experiment-to-experiment.

[0161] 4. The assay is performed by mixing the components in 12×75 mmborosilicate glass test tubes (the order of addition does not matterunless indicated) and incubating at 37° C. for 5 to 10 minutes such thatthe assay within the linear range for time and protein.

[0162] 5. The reaction is quenched by adding 1.3 ml ofchloroform/methanol/HCl=48/51/0.7 and vortexing. 10 μl of carriersolution is then added (3 mg/ml each PA, ex. 16:0-18:1, and lysoPA, exsn-1-18:1, in chloroform). Two phases are formed by adding 0.3 ml ofwater to each tube and vortexing.

[0163] 6. The sample is centrifuged for 3 minutes at 1000×g, the upper(aqueous/methanol) phase is aspirated and the lower phase is dried undernitrogen.

[0164] 7. Thin layer chromatography:

[0165] a. The dried samples are resuspended in 50 μl of chloroform and a15 μl aliquot is immediately spotted on an Analtech silica gel 60 HP-TLCplate (10×20 cm).

[0166] b. Plates are developed in chloroform/methanol/aceticacid/water=85/12.5/12.5/3 (takes about 15 min) and dried.

[0167] c. To be able to convert pixel volume (determined by the Stormphosphor imager, see step 8b) into cpm, cpm standard curve must begenerated on the plate. ¹⁴C-labeled oleate dilutions in chloroform aremade for this purpose. Four stocks (50 cpm/μl to 800 cpm/μl) are madeand 2 μl of a different concentration are spotted in each corner of theplate (where previously there was no radioactivity).

[0168] d. For quality control purposes, the plates are stained withprimuline and scanned with the Storm (blue chemilluminescence mode).

[0169] The PA and lysoPA bands are easily detected in this systembecause of the carrier added in step 5. PA and lysoPA have respectiveRf's of about 0.63 and 0.21.

[0170] 8. Quantitating Activity:

[0171] a. The plates are then wrapped in saran wrap and exposed to afreshly blanked phosphor screen overnight (longer exposures can also bedone to increase the signal).

[0172] b. The screens are scanned (Phosphorimager mode), and LPAATactivity is determined by quantifying the pixels in the band comigratingwith PA standard versus the standard curve generated from the cpmstandards that were spotted in step 7c.

[0173]FIG. 9 shows examples of a compound, namely,6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine, selected from thesecondary screening that exhibit concentration-dependent inhibition ofLPAAT-β activity (O). Moreover, these compounds have minimal effect onLPAAT-α activity ( ), suggesting they are isoform-specific inhibitors.

EXAMPLE 4

[0174] Triazines of the preferred embodiments of the present inventionwere synthesized by one of two methods. Symmetrically substitutedtriazines were synthesized by the addition of two equivalents of theappropriate amino compound, in the presence of diisopropylethylamine, tocyanuric chloride. Non-symmetrical triazines were synthesized in astepwise fashion by the sequential addition of the amino compound in thepresence of potassium carbonate with isolation of the intermediatemono-amino-dichlorotriazine.

[0175] A. Method 1: Synthesis of Symmetrical Triazines.

Synthesis of 6-Chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine(CT-113020)

[0176] To a mixture of cyanuric chloride (5.07 g, 27.5 mmol) andacetonitrile (60 ml), cooled in an ice bath, was added a solution ofaniline (5.3 ml, 58.2 mmol) and diisopropylethylamine (10.5 ml, 60.3mmol) in acetonitrile (15 ml) over 30 minutes. After stirring at roomtemperature for 20 hours, the mixture was filtered. The solid was washedwith ethyl acetate (4×25 ml), suspended in water (50 ml), stirred for 1hour, filtered, washed with water, and dried under vacuum to give6-chloro-N,N′-diphenyl-[1,3,5]triazine-2,4-diamine (3.97 g, 48% yield)as a white solid. ¹H NMR (d₆-DMSO) δ 7.61 (2 H, br s), 7.28-7.37 (4 H,m), 7.05-7.11 (4 H, m). ¹³C NMR (d₆-DMSO) δ 167.7, 164.0, 138.5, 128.4,124.5, 121.3.

[0177] B. Method 2: Synthesis of Unsymmetrical Triazines.

Synthesis of6-Chloro-N-(4-chlorophenyl)-N′-phenyl-[1,3,5]triazine-2,4-diamine(CT-116433)

[0178] To a mixture of cyanuric chloride (5.15 g, 27.9 mmol), potassiumcarbonate (3.98 g, 28.8 mmol) and 18-crown-6 (158 mg, 0.60 mmol) intoluene (40 ml), cooled in an ice bath, was added a solution of4-chloroaniline (3.61 g, 28.3 mmol) in toluene (20 ml) over 15 minutes.After stirring at room temperature for 20 hours, the mixture was treatedwith ethyl acetate (60 ml) and filtered through a pad of celite undersuction. The filtrate was concentrated under vacuum and recrystallized(chloroform) to give(4-chlorophenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine (4.06 g, 53%yield) as a white solid. ¹H NMR (d₆-DMSO) δ 11.23 (1 H, s), 7.62 (2 H,d, J=9 Hz), 7.47 (2 H, d, J=9 Hz). ¹³C NMR (d₆-DMSO) δ 169.5, 168.5,163.6, 135.7, 128.6, 122.5, ESMS m/z 273 (M−H)⁻.

[0179] To a mixture of(4-chloro-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine (3.97 g, 14.4mmol), potassium carbonate (2.20 g, 15.9 mmol) and 18-crown-6 (46 mg,0.17 mmol) in toluene (25 ml), cooled in an ice bath, was added asolution of aniline (1.4 ml, 15.4 mmol) in toluene (10 ml) over 15minutes. After stirring at room temperature for 24 hours, the mixturewas treated with ethyl acetate (35 ml) and filtered through a pad ofcelite under suction. The filtrate was concentrated under vacuum and theresidue was recrystallized (chloroform) to give6-Chloro-N-(4-chlorophenyl)-N′-phenyl-[1,3,5]triazine-2,4-diamine (2.20g, 46% yield) as a white solid. ¹H NMR (d₆-DMSO) δ 10.1-10.4 (2 H, brs), 7.5-7.8 (4 H, br s), 7.3-7.5 (4 H, m), 7.15-7.05 (1 H, m), ESMS m/z330 (M−H)⁻.

EXAMPLE 5 Synthesis of6-Chloro-N-(4-chlorophenyl)-N′-(4-methoxyphenyl)-[1,3,5]triazine-2,4-diamine(CT-31867)

[0180] The reaction of(4-chloro-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine withp-anisidine, according to method 2, gave6-chloro-N-(4-chlorophenyl)-N′-(4-methoxyphenyl)-[1,3,5]triazine-2,4-diamine(51 mg, 62%): ¹H NMR (CDCl₃) 7.21-7.50 (6 H, m), 6.91 (2 H, d, J=11 Hz),3.85 (3 H, s).

EXAMPLE 6 Synthesis of6-Chloro-N-(4-methoxyphenyl)-N′-phenyl-[1,3,5]triazine-2,4-diamine(CT-31942)

[0181] To a solution of2-(4-methoxyphenyl)amino-4,6-dichloro-1,3,5-triazine (57 mg, 0.21mmoles) in acetonitrile (0.5 ml) was added a solution of aniline (0.021ml, 0.23 mmoles) and diisopropylethylamine (0.05 ml, 0.29 mmoles) inacetonitrile (0.5 ml). After stirring for 24 hours, the mixture wasconcentrated under vacuum and the residue was dissolved in ethyl acetate(10 ml). The solution was washed with a solution composed of saturatedaqueous sodium chloride solution and 1 M hydrochloric acid (1:1, 2×10ml), dried over sodium sulfate, filtered, and concentrated under vacuum.The residue was purified by flash chromatography on silica gel elutingwith 10% ethyl acetate-hexane to provide CT-31942 (38 mg, 57% yield). ¹HNMR (d₆-DMSO) δ 10.00-10.22 (m, 2 H), 7.26-7.82 (m, 6 H), 7.01-7.10 (m,1 H), 6.91 (d, J=9 Hz,2 H), 3.74 (s,3 H).

EXAMPLE 7 Synthesis ofN-Benzo[1,3]dioxol-5-yl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine(CT-31978)

[0182] The reaction of4,6-dichloro-N-(4-chlorophenyl)-[1,3,5]triazine-2-amine with3,4-methylenedioxyaniline using the method described for the synthesisof CT-116433 provided CT-31978 (56 mg, 65% yield). ¹H NMR (d₆-DMSO) δ10.08-10.37 (m, 2 H), 7.58-7.87 (m, 2 H), 7.21-7.40 (m, 3 H), 6.85-7.03(m, 2 H), 6.00 (s, 2 H).

EXAMPLE 8 Synthesis of6-Chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N′-phenyl-[1,3,5]triazine-2,4-diamine(CT-32028)

[0183] To a solution of 4,6-dichloro-N-phenyl-[1,3,5]triazine-2-amine(95 mg, 0.39 mmoles) in tetrahydrofuran (2 ml) was added a solution of1,4-benzodioxan-6-amine (64 mg, 0.42 mmoles) and triethylamine (0.07 ml,0.50 mmoles) in tetrahydrofuran (0.5 ml). After stirring for 24 hours,the mixture was concentrated under vacuum and the residue was dissolvedin ethyl acetate (10 ml). The solution was washed with a solutioncomposed of saturated aqueous sodium chloride solution and 1 Mhydrochloric acid (1:1, 2×10 ml), dried over sodium sulfate, filtered,and concentrated under vacuum. The residue was purified by flashchromatography on silica gel eluting with 25% ethyl acetate-hexanes toprovide CT-32028 (97 mg, 69% yield). ¹H NMR (d₆-DMSO) δ 9.88-10.28 (m, 2H), 7.60-7.85 (m, 2 H), 7.20-7.40 (m, 3 H), 6.96-7.10 (m, 2 H),6.77-6.81 (m, 1 H), 4.21 (s, 4 H).

EXAMPLE 9 Synthesis ofN-Benzo[1,3]dioxol-5-yl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine(CT-32042)

[0184] The reaction of 4,6-dichloro-N-phenyl-[1,3,5]triazine-2-aminewith 3,4-methylenedioxyaniline using the method described for thesynthesis of CT-32028 provided CT-32042 (51 mg, 42% yield). ¹H NMR(d₆-DMSO) δ 10.05-10.29 (m, 2 H), 7.57-7.80 (m, 2 H), 7.21-7.42 (m, 3H), 6.80-7.13 (m, 3 H), 6.03 (s, 2 H).

EXAMPLE 10 Synthesis of6-Chloro-N-indan-5-yl-N′-phenyl-[1,3,5]triazine-2,4-diamine (CT-32099)

[0185] The reaction of 4,6-dichloro-N-phenyl-[1,3,5]triazine-2-aminewith 5-aminoindan using the method described for the synthesis ofCT-32028 provided CT-32099 (36 mg, 37% yield). ¹H NMR (d₆-DMSO) δ10.15-10.28 (m, 2 H), 7.56-7.72 (m, 3 H), 7.05-7.39 (m, 5 H), 2.75-90(m, 4 H), 1.94-2.09 (m, 2 H).

EXAMPLE 11 Synthesis of6-chloro-N-2-(4-chlorophenyl)-N-4-propyl-[1,3,5]triazine-2,4-diamine(CT-116988)

[0186] The reaction of4,6-dichloro-N-(4-chlorophenyl)-[1,3,5]triazine-2-amine with propylamineusing the method described for the synthesis of CT-116433 providesCT-116988.

EXAMPLE 12 Synthesis ofN-(4-Chlorophenyl)-6-methoxy-N′-propyl-[1,3,5]triazine-2,4-diamine(CT-117147)

[0187] A mixture of CT-116988 and sodium methoxide (3.0 molarequivalents) in methanol is heated at reflux for 18 hours. After coolingto room temperature, the reaction mixture is concentrated under vacuum.The residue is suspended in ethyl acetate and washed with water. Theethyl acetate phase is dried over sodium sulfate, filtered andconcentrated under vacuum. The residue is purified by flashchromatography on silica gel to provide CT-117147.

EXAMPLE 13 Synthesis ofN-(4-chlorophenyl)-6-methylsulfanyl-N′-phenyl-[1,3,5]triazine-2,4-diamine(CT-31888)

[0188] A mixture of CT-116433 (108 mg, 0.32 mmol) and sodiummethanethiolate (79 mg, 1.13 mmol) in dimethyl sulfoxide (3 ml) washeated at 70° C. for 18 hours. After cooling to room temperature themixture was diluted with ethyl acetate (25 ml) and was washed with a 1:1solution of water and saturated aqueous sodium chloride solution (4×25ml). The organic phase was dried over sodium sulfate, filtered, andconcentrated under vacuum. The residue was purified by flashchromatography on silica gel eluting with 5% ethyl acetate-hexane toprovide CT-31888 (76 mg, 69% yield). ¹H NMR (d₆-DMSO) δ 7.51-7.60 (m, 4H), 7.26-7.39 (m, 5 H), 7.00-7.17 (m, 2 H), 2.56 (s, 3 H).

[0189] From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated by referencein their entirety.

What is claimed is:
 1. A compound of the Formula:

wherein, R¹ is halo, hydroxy, alkylmercapto, mercapto, alkoxy, aryloxyor substituted amino; R², R³, R⁴ and R⁵, each of which may be same ordifferent, are hydrogen, alkyl, substituted alkyl, alkenyl, alkynyl,aryl or substituted aryl; or R² and R³ or R⁴ and R⁵, together with thenitrogen to which they are attached, form a piperidine, piperazine, or amorpholine ring; or pharmaceutically acceptable salts thereof.
 2. Acompound of claim 1, wherein R¹ is chloro, R² and R⁴ are hydrogen and R³and R⁵ are phenyl; or pharmaceutically acceptable salts thereof.
 3. Acompound of claim 1, wherein R¹ is chloro, R² and R⁴ are hydrogen, R³ isphenyl and R⁵ is 4-chlorophenyl; or pharmaceutically acceptable saltsthereof.
 4. A compound of claim 1, wherein R¹ is chloro, R² and R⁴ arehydrogen, R³ is t-butyl and R⁵ is 4-chlorophenyl; or pharmaceuticallyacceptable salts thereof.
 5. A compound selected from the groupconsisting of6-chloro-N-(4-methoxy-phenyl)-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,N-butyl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-isopropyl-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,N-tert-butyl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine,(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-naphthalen-1-yl-amine,N-tert-butyl-6-chloro-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-cyclohexyl-N′-isopropyl-[1,3,5]triazine-2,4-diamine,2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-2-methyl-propan-1-ol,6-chloro-N-isopropyl-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chloro-phenyl)-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine,N-allyl-6-chloro-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine,2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-ethanol,N-tert-butyl-6-chloro-N′-cyclopentyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-methoxyphenyl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-indan-5-yl-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chloro-phenyl)-N′-propyl-[1,3,5]triazine-2,4-diamine,N-(4-chloro-phenyl)-6-methoxy-N′-propyl-[1,3,5]triazine-2,4-diamine andN-(4-chloro-phenyl)-6-methylsulfanyl-N′-phenyl-[1,3,5]triazine-2,4-diamine.6. A compound of claim 1, wherein R¹ is chloro, R² and R⁴ are hydrogen,R³ is 4-methoxyphenyl and R⁵ is 4-chlorophenyl; or pharmaceuticallyacceptable salts thereof.
 7. A pharmaceutical composition comprising thecompound of claim 1 and a pharmaceutically acceptable carrier.
 8. Amethod for inhibiting LPAAT-β (lysophosphatidic acid acyltransferase β)comprising contacting LPAAT-β with an effective amount of a compound ofthe Formula:

wherein, R¹ is halo, hydroxy, alkylmercapto, mercapto, alkoxy, aryloxyor substituted amino; R², R³, R⁴ and R⁵, each of which may be same ordifferent, are hydrogen, alkyl, substituted alkyl, alkenyl, alkynyl,aryl or substituted aryl; or R² and R³ or R⁴ and R⁵, together with thenitrogen to which they are attached, form a piperidine, piperazine, or amorpholine ring; or pharmaceutically acceptable salts thereof; therebyinhibiting LPAAT-β.
 9. The method of claim 8, wherein said LPAAT-β isfound in an animal.
 10. The method of claim 9, wherein said animal is amammal.
 11. The method of claim 10, wherein said mammal is a human. 12.The method of claim 8, wherein R¹ is chloro, R² and R⁴ are hydrogen andR³ and R⁵ are phenyl; or pharmaceutically acceptable salts thereof. 13.The method of claim 8, wherein R¹ is chloro, R² and R⁴ are hydrogen, R³is phenyl and R⁵ is 4-chlorophenyl; or pharmaceutically acceptable saltsthereof.
 14. The method of claim 8, wherein R¹ is chloro, R² and R⁴ arehydrogen, R³ is t-butyl and R⁵ is 4-chlorophenyl; or pharmaceuticallyacceptable salts thereof.
 15. The method of claim 8, wherein R¹ ischloro, R² and R⁴ are hydrogen, R³ is 4-methoxyphenyl and R⁵ is4-chlorophenyl; or pharmaceutically acceptable salts thereof.
 16. Themethod of claim 8, wherein the compound is selected from the groupconsisting of6-chloro-N-(4-methoxy-phenyl)-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,N-butyl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-isopropyl-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,N-tert-butyl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine,(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-naphthalen-1-yl-amine,N-tert-butyl-6-chloro-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-cyclo-hexyl-N′-isopropyl-[1,3,5]triazine-2,4-diamine,2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-2-methyl-propan-1-ol,6-chloro-N-isopropyl-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chloro-phenyl)-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine,N-allyl-6-chloro-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine,2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-ethanol,N-tert-butyl-6-chloro-N′-cyclopentyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-methoxyphenyl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-indan-5-yl-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chloro-phenyl)-N′-propyl-[1,3,5]triazine-2,4-diamine,N-(4-chloro-phenyl)-6-methoxy-N′-propyl-[1,3,5]triazine-2,4-diamine andN-(4-chloro-phenyl)-6-methylsulfanyl-N′-phenyl-[1,3,5]triazine-2,4-diamine.17. A method of inhibiting cell proliferation comprising contacting acell with an effective amount of a compound of the Formula:

wherein, R¹ is halo, hydroxy, alkylmercapto, mercapto, alkoxy, arylox orsubstituted amino; R², R³, R⁴ and R⁵, each of which may be same ordifferent, are hydrogen, alkyl, substituted alkyl, alkenyl, alkynyl,aryl or substituted aryl; or R² and R³ or R⁴ and R⁵, together with thenitrogen to which they are attached, form a piperidine, piperazine, or amorpholine ring; or pharmaceutically acceptable salts thereof, therebyinhibiting the proliferation of the cell.
 18. The method of claim 17,wherein said cell is a cancer cell.
 19. The method of claim 17, whereinR¹ is chloro, R² and R⁴ are hydrogen and R³ and R⁵ are phenyl; orpharmaceutically acceptable salts thereof.
 20. The method of claim 17,wherein R¹ is chloro, R² and R⁴ are hydrogen, R³ is t-butyl and R⁵ is4-chlorophenyl; or pharmaceutically acceptable salts thereof.
 21. Themethod of claim 17, wherein R¹ is chloro, R² and R⁴ are hydrogen, R³ ist-butyl and R⁵ is 4-chlorophenyl; or pharmaceutically acceptable saltsthereof.
 22. The method of claim 17, wherein R¹ is chloro, R² and R⁴ arehydrogen, R³ is 4-methoxyphenyl and R⁵ is 4-chlorophenyl; orpharmaceutically acceptable salts thereof.
 23. The method of claim 17,wherein the compound is selected from the group consisting of6-chloro-N-(4-methoxy-phenyl)-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,N-butyl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-isopropyl-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,N-tert-butyl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine,(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-naphthalen-1-yl-amine,N-tert-butyl-6-chloro-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-cyclo-hexyl-N′-isopropyl-[1,3,5]triazine-2,4-diamine,2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-2-methyl-propan-1-ol,6-chloro-N-isopropyl-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chloro-phenyl)-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine,N-allyl-6-chloro-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine,2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-ethanol,N-tert-butyl-6-chloro-N′-cyclopentyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-methoxyphenyl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-indan-5-yl-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chloro-phenyl)-N′-propyl-[1,3,5]triazine-2,4-diamine,N-(4-chloro-phenyl)-6-methoxy-N′-propyl-[1,3,5]triazine-2,4-diamine andN-(4-chloro-phenyl)-6-methylsulfanyl-N′-phenyl-[1,3,5]triazine-2,4-diamine.triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamineand 6-chloro-N-indan-5-yl-N′-phenyl-[1,3,5]triazine-2,4-diamine.
 24. Amethod for treating cancer, comprising administering to an animal inneed thereof, an effective amount of a compound of the Formula:

wherein, R¹ is halo, hydroxy, alkylmercapto, mercapto, alkoxy, aryloxyor substituted amino; R², R³, R⁴ and R⁵, each of which may be same ordifferent, are hydrogen, alkyl, substituted alkyl, alkenyl, alkynyl,aryl or substituted aryl; or R² and R³ or R⁴ and R⁵, together with thenitrogen to which they are attached, form a piperidine, piperazine, or amorpholine ring; or pharmaceutically acceptable salts thereof; whereinthe cancer is treated.
 25. The method of claim 24, wherein R¹ is chloro,R² and R⁴ are hydrogen and R³ and R⁵ are phenyl; or pharmaceuticallyacceptable salts thereof.
 26. The method of claim 24, wherein R¹ ischloro, R² and R⁴ are hydrogen, R³ is t-butyl and R⁵ is 4-chlorophenyl;or pharmaceutically acceptable salts thereof.
 27. The method of claim24, wherein R¹ is chloro, R² and R⁴ are hydrogen, R³ is t-butyl and R⁵is 4-chlorophenyl; or pharmaceutically acceptable salts thereof.
 28. Themethod of claim 24, wherein R¹ is chloro, R² and R⁴ are hydrogen, R³ is4-methoxyphenyl and R⁵ is 4-chlorophenyl; or pharmaceutically acceptablesalts thereof.
 29. The method of claim 24, wherein said cancer isprostate, breast, lung, ovarian, brain, cervical, colon or bladdercancer.
 30. The method of claim 24, where the compound is selected fromthe group consisting of6-chloro-N-(4-methoxy-phenyl)-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,N-butyl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-isopropyl-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,N-tert-butyl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine,(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-naphthalen-1-yl-amine,N-tert-butyl-6-chloro-N′-p-tolyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-cyclo-hexyl-N′-isopropyl-[1,3,5]triazine-2,4-diamine,2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-2-methyl-propan-1-ol,6-chloro-N-isopropyl-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chloro-phenyl)-N-cyclohexyl-[1,3,5]triazine-2,4-diamine,N-allyl-6-chloro-N′-cyclohexyl-[1,3,5]triazine-2,4-diamine,2-(4-chloro-6-phenylamino-[1,3,5]triazin-2-ylamino)-ethanol,N-tert-butyl-6-chloro-N′-cyclopentyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-methoxyphenyl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-(4-chlorophenyl)-[1,3,5]triazine-2,4-diamine,6-chloro-N-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-N′-phenyl-[1,3,5]triazine-2,4-diamine,N-benzo[1,3]dioxol-5-yl-6-chloro-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-indan-5-yl-N′-phenyl-[1,3,5]triazine-2,4-diamine,6-chloro-N-(4-chloro-phenyl)-N′-propyl-[1,3,5]triazine-2,4-diamine,N-(4-chloro-phenyl)-6-methoxy-N′-propyl-[1,3,5]triazine-2,4-diamine andN-(4-chloro-phenyl)-6-methylsulfanyl-N′-phenyl-[1,3,5]triazine-2,4-diamine.31. A method for screening a patient for LPAAT-β activity, said methodcomprising detecting the presence or absence of an increased amount ofLPAAT-β RNA, DNA or protein relative to a predetermined control, wherebythe presence of said increased amount is indicative of cancersusceptibility in said patient.
 32. The method of claim 31, comprisingdetecting the presence or absence of an increased amount of LPAAT-β RNA.33. The method of claim 31, comprising detecting the presence or absenceof an increased amount of LPAAT-β DNA.
 34. The method of claim 31,comprising detecting the presence or absence of an increased amount ofLPAAT-β protein.
 35. A method of inhibiting cell proliferationcomprising the inhibition of LPAAT-β.
 36. The method of claim 35,wherein said cell is a cancer cell.
 37. A vaccine preparation capable ofinducing an anti-tumor immune response comprising a pharmaceuticallyacceptable carrier and an anti-tumor immune response-inducing effectiveamount of LPAAT-β protein.
 38. A method for screening a patient forLPAAT-β activity, said method comprising detecting the presence orabsence of an increased amount of a phospholipid of defined acyl-chaincomposition relative to a predetermined control, whereby the presence ofsaid increased amount is indicative of cancer susceptibility in saidpatient.
 39. The method of claim 38, wherein said phospholipid isphosphatidylinositol.